1.1 Field of the Invention
Disclosed are genetically engineered, recombinant .delta.-endotoxin polypeptide compositions derived from Bacillus thuringiensis that are useful in the control of Coleopteran insects such as Colorado Potato Beetle (Leptinotarsa decemlineata), southern corn rootworm (Diabrotica undecimpunctata howardi Barber) and western corn rootworm (Diabrotica virgifera virgifera LeConte). Also disclosed are methods of preparing and using these polypeptides in insecticidal formulations for killing insect pests.
1.2 Description of the Related Art
Almost all field crops, plants, and commercial farming areas are susceptible to attack by one or more insect pests. Particularly problematic are Coleopteran and Lepidoptern pests. For example, vegetable and cole crops such as artichokes, kohlrabi, arugula, leeks, asparagus, lentils, beans, lettuce (e.g., head, leaf, romaine), beets, bok choy, malanga, broccoli, melons (e.g., muskmelon, watermelon, crenshaw, honeydew, cantaloupe), brussels sprouts, cabbage, cardoni, carrots, napa, cauliflower, okra, onions, celery, parsley, chick peas, parsnips, chicory, peas, chinese cabbage, peppers, collards, potatoes, cucumber, pumpkins, cucurbits, radishes, dry bulb onions, rutabaga, eggplant, salsify, escarole, shallots, endive, soybean, garlic, spinach, green onions, squash, greens, sugar beets, sweet potatoes, turnip, swiss chard, horseradish, tomatoes, kale, turnips, and a variety of spices are sensitive to infestation by one or more of the following insect pests: alfalfa looper, armyworm, beet armyworm, artichoke plume moth, cabbage budworm, cabbage looper, cabbage webworm, corn earworm, celery leafeater, cross-striped cabbageworm, european corn borer, diamondback moth, green cloverworm, imported cabbageworm, melonworm, omnivorous leafroller, pickleworm, rindworm complex, saltmarsh caterpillar, soybean looper, tobacco budworm, tomato fruitworm, tomato hornworm, tomato pinworm, velvetbean caterpillar, and yellowstriped armyworm. Likewise, pasture and hay crops such as alfalfa, pasture grasses and silage are often attacked by such pests as armyworm, beef armyworm, alfalfa caterpillar, European skipper, a variety of loopers and webworms, as well as yellowstriped armyworms.
Fruit and vine crops such as apples, apricots, cherries, nectarines, peaches, pears, plums, prunes, quince almonds, chestnuts, filberts, pecans, pistachios, walnuts, citrus, blackberries, blueberries, boysenberries, cranberries, currants, loganberries, raspberries, strawberries, grapes, avocados, bananas, kiwi, persimmons, pomegranate, pineapple, tropical fruits are often susceptible to attack and defoliation by achema sphinx moth, amorbia, armyworm, citrus cutworm, banana skipper, blackheaded fireworm, blueberry leafroller, cankerworm, cherry fruitworm, citrus cutworm, cranberry girdler, eastern tent caterpillar, fall webworm, fall webworm, filbert leafroller, filbert webworm, fruit tree leafroller, grape berry moth, grape leaffolder, grapeleaf skeletonizer, green fruitworm, gummosos-batrachedra commosae, gypsy moth, hickory shuckworm, hornworms, loopers, navel orangeworm, obliquebanded leafroller, omnivorous leafroller. omnivorous looper, orange tortrix, orangedog, oriental fruit moth, pandemis leafroller, peach twig borer, pecan nut casebearer, redbanded leafroller, redhumped caterpillar, roughskinned cutworm, saltmarsh caterpillar, spanworm, tent caterpillar, thecla-thecla basillides, tobacco budworm, tortrix moth, tufted apple budmoth, variegated leafroller, walnut caterpillar, western tent caterpillar, and yellowstriped armyworm.
Field crops such as canola/rape seed, evening primrose, meadow foam, corn (field, sweet, popcorn), cotton, hops, jojoba, peanuts, rice, safflower, small grains (barley, oats, rye, wheat, etc.), sorghum, soybeans, sunflowers, and tobacco are often targets for infestation by insects including armyworm, asian and other corn borers, banded sunflower moth, beet armyworm, bollworm, cabbage looper, corn rootworm (including southern and western varieties), cotton leaf perforator, diamondback moth, european corn borer, green cloverworm, headmoth, headworm, imported cabbageworm, loopers (including Anacamptodes spp.), obliquebanded leafroller, omnivorous leaftier, podworm, podworm, saltmarsh caterpillar, southwestern corn borer, soybean looper, spotted cutworm, sunflower moth, tobacco budworm, tobacco hornworm, velvetbean caterpillar, Bedding plants, flowers, ornamentals, vegetables and container stock are frequently fed upon by a host of insect pests such as armyworm, azalea moth, beet armyworm, diamondback moth, ello moth (hornworm), Florida fern caterpillar, Io moth, loopers, oleander moth, omnivorous leafroller, omnivorous looper, and tobacco budworm.
Forests, fruit, ornamental, and nut-bearing trees, as well as shrubs and other nursery stock are often susceptible to attack from diverse insects such as bagworm, blackheaded budworm, browntail moth, California oakworm, douglas fir tussock moth, elm spanworm, fall webworm, fruittree leafroller, greenstriped mapleworm, gypsy moth, jack pine budworm, mimosa webworm, pine butterfly, redhumped caterpillar, saddleback caterpillar, saddle prominent caterpillar, spring and fall cankerworm, spruce budworm, tent caterpillar, tortrix, and western tussock moth. Likewise, turf grasses are often attacked by pests such as armyworm, sod webworm, and tropical sod webworm.
Because crops of commercial interest are often the target of insect attack, environmentally-sensitive methods for controlling or eradicating insect infestation are desirable in many instances. This is particularly true for farmers, nurserymen, growers, and commercial and residential areas which seek to control insect populations using eco-friendly compositions.
The most widely used environmentally-sensitive insecticidal formulations developed in recent years have been composed of microbial pesticides derived from the bacterium Bacillus thuringiensis. B. thuringiensis is a Gram-positive bacterium that produces crystal proteins or inclusion bodies which are specifically toxic to certain orders and species of insects. Many different strains of B. thuringiensis have been shown to produce insecticidal crystal proteins. Compositions including B. thuringiensis strains which produce insecticidal proteins have been commercially-available and used as environmentally-acceptable insecticides because they are quite toxic to the specific target insect, but are harmless to plants and other non-targeted organisms.
1.2.1 .delta.-Endotoxins
.delta.-endotoxins are used to control a wide range of leaf-eating caterpillars and beetles, as well as mosquitoes. These proteinaceous parasporal crystals, also referred to as insecticidal crystal proteins, crystal proteins, Bt inclusions, crystaline inclusions, inclusion bodies, and Bt toxins, are a large collection of insecticidal proteins produced by B. thuringiensis that are toxic upon ingestion by a susceptible insect host. Over the past decade research on the structure and function of B. thuringiensis toxins has covered all of the major toxin categories, and while these toxins differ in specific structure and function, general similarities in the structure and function are assumed. Based on the accumulated knowledge of B. thuringiensis toxins, a generalized mode of action for B. thuringiensis toxins has been created and includes: ingestion by the insect, solubilization in the insect midgut (a combination stomach and small intestine), resistance to digestive enzymes sometimes with partial digestion actually "activating" the toxin, binding to the midgut cells, formation of a pore in the insect cells and the disruption of cellular homeostasis (English and Slatin, 1992).
1.2.2 Genes Encoding Crystal Proteins
Many of the .delta.-endotoxins are related to various degrees by similarities in their amino acid sequences. Historically, the proteins and the genes which encode them were classified based largely upon their spectrum of insecticidal activity. The review by Hofte and Whiteley (1989) discusses the genes and proteins that were identified in B. thuringiensis prior to 1990, and sets forth the nomenclature and classification scheme which has traditionally been applied to B. thuringiensis genes and proteins. cryI genes encode lepidopteran-toxic CryI proteins. cryII genes encode CryII proteins that are toxic to both lepidopterans and dipterans. cryIII genes encode coleopteran-toxic CryIII proteins, while cryIV genes encode dipteran-toxic CryIV proteins.
Based on the degree of sequence similarity, the proteins were further classified into subfamilies; more highly related proteins within each family were assigned divisional letters such as CryIA, CryIB, CryIC, etc. Even more closely related proteins within each division were given names such as CryIC1, CryIC2, etc.
Recently a new nomenclature was developed which systematically classified the Cry proteins based upon amino acid sequence homology rather than upon insect target specificities. The classification scheme for many known toxins, not including allelic variations in individual proteins, is summarized in Table 1.
TABLE 1 ______________________________________ KNOWN B. THURINGIENSIS .delta.-ENDOTOXINS, GENBANK ACCESSION NUMBERS, AND REVISED NOMENCLATURE.sup.A GenBank New Old Accession # ______________________________________ Cry1Aa1 CryIA(a) M11250 Cry1Aa2 CryIA(a) M10917 Cry1Aa3 CryIA(a) D00348 Cry1Aa4 CryIA(a) X13535 Cry1Aa5 CryIA(a) D175182 Cry1Aa6 CryIA(a) U43605 Cry1Ab1 CryIA(b) M13898 Cry1Ab2 CryIA(b) M12661 Cry1Ab3 CryIA(b) M15271 Cry1Ab4 CryIA(b) D00117 Cry1Ab5 CryIA(b) X04698 Cry1Ab6 CryIA(b) M37263 Cry1Ab7 CryIA(b) X13233 Cry1Ab8 CryIA(b) M16463 Cry1Ab9 CryIA(b) X54939 Cry1Ab10 CryIA(b) A29125 Cry1Ac1 CryIA(c) M11068 Cry1Ac2 CryIA(c) M35524 Cry1Ac3 CryIA(c) X54159 Cry1Ac4 CryIA(c) M73249 Cry1Ac5 CryIA(c) M73248 Cry1Ac6 CryIA(c) U43606 Cry1Ac7 CryIA(c) U87793 Cry1Ac8 CryIA(c) U87397 Cry1Ac9 CryIA(c) U89872 Cry1Ac10 CryIA(c) AJ002514 Cry1Ad1 CryIA(d) M73250 Cry1Ae1 CryIA(e) M65252 Cry1Ba1 CryIB X06711 Cry1Ba2 X95704 Cry1Bb1 ET5 L32020 Cry1Bc1 CryIb(c) Z46442 Cry1Bd1 CryB1 Cry1Ca1 CryIC X07518 Cry1Ca2 CryIC X13620 Cry1Ca3 CryIC M73251 Cry1Ca4 CryIC A27642 Cry1Ca5 CryIC X96682 Cry1Ca6 CryIC X96683 Cry1Ca7 CryIC X96684 Cry1Cb1 CryIC(b) M97880 Cry1Da1 CryID X54160 Cry1Db1 PrtB Z22511 Cry1Ea1 CryIE X53985 Cry1Ea2 CtyIE X56144 Cry1Ea3 CryIE M73252 Cry1Ea4 U94323 Cry1Eb1 CryIE(b) M73253 Cry1Fa1 CryIF M63897 Cry1Fa2 CryIF M63897 Cry1Fb1 PrtD Z22512 Cry10a1 PrtA Z22510 Cry1Ga2 CryIM Y09326 Cry1Gb1 CryH2 Cry1Ha1 PrtC Z22513 Cry1Hb1 U35780 Cry1Ia1 CryV X62821 Cry1Ia2 CryV M98544 Cry1Ia3 CryV L36338 Cry1Ia4 CryV M49391 Cry1Ia5 CryV Y08920 Cry1Ib1 CryV U07642 Cry1Ja1 ET4 L32019 Cry1Jb1 ET1 U31527 Cry1Ka1 U28801 Cry2Aa1 CryIIA M31738 Cry2Aa2 CryIIA M23723 Cry2Aa3 D86084 Cry2Ab1 CryIIB M23724 Cry2Ab2 CryIIB X55416 Cry2Ac1 CryIIC X57252 Cry3Aa1 CryIIIA M22472 Cry3Aa2 CryIIIA J02978 Cry3Aa3 CryIIIA Y00420 Cry3Aa4 CryIIIA M30503 Cry3Aa5 CryIIIA M37207 Cry3Aa6 CryIIIA U10985 Cry3Ba1 CryIIIB X17123 Cry3Ba2 CryIIIB A07234 Cry3Bb1 CryIIIB2 M89794 Cry3Bb2 CryIIIC(b) U31633 Cry3Ca1 CryIIID X59797 Cry4Aa1 CryIVA Y00423 Cry4Aa2 CryIVA D00248 Cry4Ba1 CryIVB X07423 Cry4Ba2 CryIVB X07082 Cry4Ba3 CryIVB M20242 Cry4Ba4 CryIVB D00247 Cry5Aa1 CryVA(a) L07025 Cry5Ab1 CryVA(b) L07026 Cry5Ba1 PS86Q3 U19725 Cry6Aa1 CryVIA L07022 Cry6Ba1 CryVIB L07024 Cry7Aa1 CryIIIC M64478 Cry7Ab1 CryIIICb U04367 Cry8Aa1 CryIIIE U04364 Cry8Ba1 CryIIIG U04365 Cry8Ca1 CryIIIF U04366 Cry9Aa1 CryIG X58120 Cry9Aa2 CryIG X58534 Cry9Ba1 CryIX X75019 Cry9Ca1 CryIH Z37527 Cry9Da1 N141 D85560 Cry10Aa1 CryIVC M12662 Cry11Aa1 CryIVD M31737 Cry11Aa2 CryIVD M22860 Cry11Ba1 Jeg80 X86902 Cry12Aa1 CryVB L07027 Cry13Aa1 CryVC L07023 Cry14Aa1 CryVD U13955 Cry15Aa1 34kDa M76442 Cry16Aa1 cbm71 X94146 Cry17Aa1 cbm71 X99478 Cry18Aa1 CryBP1 X99049 Cry19Aa1 Jeg65 Y08920 Cry20Aa1 U82518 Cry21Aa1 I32932 Cry22Aa1 I34547 Cyt1Aa1 CytA X03182 Cyt1Aa2 CytA X04338 Cyt1Aa3 CytA Y00135 Cyt1Aa4 CytA M35968 Cyt1Ab1 CytM X98793 Cyt1Ba1 U37196 Cyt2Aa1 CytB Z14147 Cyt2Ba1 "CytB" U52043 Cyt2Ba2 "CytB" AF020789 Cyt2Ba3 "CytB" AF022884 Cyt2Ba4 "CytB" AF022885 Cyt2Ba5 "CytB" AF022886 Cyt2Bb1 U82519 ______________________________________ .sup.A Adapted from: http://epunix.biols.susx.ac.uk/Home/Neil.sub.-- Crickmore/Bt/index.html
1.2.3 Bioinsecticide Polypeptide Compositions
The utility of bacterial crystal proteins as insecticides was extended beyond lepidopterans and dipteran larvae when the first isolation of a coleopteran-toxic B. thuringiensis strain was reported (Krieg et al., 1983; 1984). This strain (described in U.S. Pat. No. 4,766,203, specifically incorporated herein by reference), designated B. thuringiensis var. tenebrionis, is reported to be toxic to larvae of the coleopteran insects Agelastica alni (blue alder leaf beetle) and Leptinotarsa decemlineata (Colorado potato beetle).
U.S. Pat. No. 5,024, 837 also describes hybrid B. thuringiensis var. kurstaki strains which showed activity against lepidopteran insects. U.S. Pat. No. 4,797,279 (corresponding to EP 0221024) discloses a hybrid B. thuringiensis containing a plasmid from B. thuringiensis var. kurstaki encoding a lepidopteran-toxic crystal protein-encoding gene and a plasmid from B. thuringiensis tenebrionis encoding a coleopteran-toxic crystal protein-encoding gene. The hybrid B. thuringiensis strain produces crystal proteins characteristic of those made by both B. thuringiensis kurstaki and B. thuringiensis tenebrionis. U.S. Pat. No. 4,910,016 (corresponding to EP 0303379) discloses a B. thuringiensis isolate identified as B. thuringiensis MT 104 which has insecticidal activity against coleopterans and lepidopterans.
1.2.4 Molecular Genetic Techniques Facilitate Protein Engineering
The revolution in molecular genetics over the past decade has facilitated a logical and orderly approach to engineering proteins with improved properties. Site specific and random mutagenesis methods, the advent of polymerase chain reaction (PCR.TM.) methodologies, and related advances in the field have permitted an extensive collection of tools for changing both amino acid sequence, and underlying genetic sequences for a variety of proteins of commercial, medical, and agricultural interest.
Following the rapid increase in the number and types of crystal proteins which have been identified in the past decade, researchers began to theorize about using such techniques to improve the insecticidal activity of various crystal proteins. In theory, improvements to .delta.-endotoxins should be possible using the methods available to protein engineers working in the art, and it was logical to assume that it would be possible to isolate improved variants of the wild-type crystal proteins isolated to date. By strengthening one or more of the aforementioned steps in the mode of action of the toxin, improved molecules should provide enhanced activity, and therefore, represent a breakthrough in the field. If specific amino acid residues on the protein are identified to be responsible for a specific step in the mode of action, then these residues can be targeted for mutagenesis to improve performance
1.2.5 Structural Analyses of Crystal Proteins
The combination of structural analyses of B. thuringiensis toxins followed by an investigation of the function of such structures, motifs, and the like has taught that specific regions of crystal protein endotoxins are, in a general way, responsible for particular functions.
Domain 1, for example, from Cry3Bb and Cry1Ac has been found to be responsible for ion channel activity, the initial step in formation of a pore (Walters et al., 1993; Von Tersch et al., 1994). Domains 2 and 3 have been found to be responsible for receptor binding and insecticidal specificity (Aronson et al., 1995; Caramori et al., 1991; Chen et al. 1993; de Maagd et al., 1996; Ge et al., 1991; Lee et al., 1992; Lee et al., 1995; Lu et al., 1994; Smedley and Ellar, 1996; Smith and Ellar, 1994; Rajamohan et al., 1995; Rajamohan et al., 1996; Wu and Dean, 1996). Regions in domain 2 and 3 can also impact the ion channel activity of some toxins (Chen et al., 1993, Wolfersberger et al., 1996; Von Tersch et al., 1994).
1.3 Deficiencies in the Prior Art
Unfortunately, while many laboratories have attempted to make mutated crystal proteins, few have succeeded in making mutated crystal proteins with improved lepidopteran toxicity. In almost all of the examples of genetically-engineered B. thuringiensis toxins in the literature, the biological activity of the mutated crystal protein is no better than that of the wild-type protein, and in many cases, the activity is decreased or destroyed altogether (Almond and Dean, 1993; Aronson et al., 1995; Chen et al., 1993, Chen et al., 1995; Ge et al., 1991; Kwak et al., 1995; Lu et al., 1994; Rajamohan et al., 1995; Rajamohan et al., 1996; Smedley and Ellar, 1996; Smith and Ellar, 1994; Wolfersberger et al., 1996; Wu and Aronson, 1992).
For a crystal protein having approximately 650 amino acids in the sequence of its active toxin, and the possibility of 20 different amino acids at each position in this sequence, the likelihood of arbitrarily creating a successful new structure is remote, even if a general function to a stretch of 250-300 amino acids can be assigned. Indeed, the above prior art with respect to crystal protein gene mutagenesis has been concerned primarily with studying the structure and function of the crystal proteins, using mutagenesis to perturb some step in the mode of action, rather than with engineering improved toxins.
Collectively, the limited successes in the art to develop synthetic toxins with improved insecticidal activity have stifled progress in this area and confounded the search for improved endotoxins or crystal proteins. Rather than following simple and predictable rules, the successful engineering of an improved crystal protein may involve different strategies, depending on the crystal protein being improved and the insect pests being targeted. Thus, the process is highly empirical.
Accordingly, traditional recombinant DNA technology is clearly not routine experimentation for providing improved insecticidal crystal proteins. What are lacking in the prior art are rational methods for producing genetically-engineered B. thuringiensis crystal proteins that have improved insecticidal activity and, in particular, improved toxicity towards a wide range of lepidopteran insect pests.